LEC1 (NF-YB9) directly interacts with LEC2 to control gene expression in seed.

The LAFL transcription factors LEC2, ABI3, FUS3 and LEC1 are master regulators of seed development. LEC2, ABI3 and FUS3 are closely related proteins that contain a B3-type DNA binding domain. We have previously shown that LEC1 (a NF-YB type protein) can increase LEC2 and ABI3 but not FUS3 activity. Interestingly, FUS3, LEC2 and ABI3 contain a B2 domain, the function of which remains elusive. We showed that LEC1 and LEC2 partially co-localised in the nucleus of developing embryos. By comparing protein sequences from various species, we identified within the B2 domains a set of highly conserved residues (i.e. TKxxARxxRxxAxxR). This domain directly interacts with LEC1 in yeast. Mutations of the conserved amino acids of the motif in the B2 domain abolished this interaction both in yeast and in moss protoplasts and did not alter the nuclear localisation of LEC2 in planta. Conversely, the mutations of key amino acids for the function of LEC1 in planta (D86K) prevented the interaction with LEC2. These results provide molecular evidences for the binding of LEC1 to B2-domain containing transcription factors, to form heteromers, involved in the control of gene expression.

Apolipoprotein E-genotype dependent hippocampal and cortical responses to traumatic brain injury.

The different alleles of the apolipoprotein E gene (APOE-gene, ApoE-protein) have been reported to influence recovery after traumatic brain injury (TBI) in both human patients and animal models, with the e4 allele typically conferring poorer prognosis for recovery. How the E4 allele, and consequently the ApoE4 isoform, affects recovery is unknown, but proposed mechanisms include neurogenesis, inflammatory response and amyloid processing or metabolism. Using the controlled cortical impact (CCI) model of brain injury and microarray technology we have characterized the genomic response to injury in the brains of APOE2, APOE3 and APOE4 transgenic mice and identified quantitatively and qualitatively significantly different profiles of gene expression in both the hippocampus and the cortex of the APOE3 mice compared to APOE4. The observed gene regulation predicts functional consequences including effects on inflammatory processes, cell growth and proliferation, and cellular signaling, and may suggest that the poor recovery post-TBI in APOE4 animals and human patients is less likely to result from a specific activation of neurodegenerative mechanisms than a loss of reparative capability.

The role of calcium and magnesium ions in uptake of beta-amyloid peptides by microglial cells.

Amyloid peptides 1-40 and 1-42 (Abeta 1-40 and Abeta 1-42) are major components of diffuse and neuritic senile plaques present in the brain of patients with Alzheimers disease. Their interaction with microglial cells was studied using a system partly mimicking these plaques, which consisted in heat-killed yeast particles coated with either Abeta 1-40 or Abeta 1-42. Using these particles, it has been shown in our laboratory that LRP is involved mainly in the elimination of Abeta 1-42-coated heat-killed yeast particles and partly in that of Abeta 1-40-coated heat-killed yeast particles by microglial cells in culture. We show here that in the presence of calcium and magnesium ions extracellular chelators, namely EDTA (for both ions) and EGTA (for calcium ions), the internalization of coated heat-killed particles was impaired. In the presence of BAPTA-AM, an intracellular chelator of calcium ions and thapsigargin, an inhibitor of the endoplasmic reticulum calcium pump, no effect was observed on the phagocytosis of Abeta 1-40-coated heat-killed yeast particles, whereas that of Abeta 1-42-coated heat-killed yeast particles was affected. These results suggest that different signaling mechanisms are involved after the internalization of Abeta 1-40 and Abeta 1-42.

Indirect evidence from DNA sequence diversity for genetic degeneration of the Y-chromosome in dioecious species of the plant Silene: the SlY4/SlX4 and DD44-X/DD44-Y gene pairs.

The action of natural selection is expected to reduce the effective population size of a nonrecombining chromosome, and this is thought to be the chief factor leading to genetic degeneration of Y-chromosomes, which cease recombining during their evolution from ordinary chromosomes. Low effective population size of Y chromosomes can be tested by studying DNA sequence diversity of Y-linked genes. In the dioecious plant, Silene latifolia, which has sex chromosomes, one comparison (SlX1 vs. SlY1) indeed finds lower Y diversity compared with the homologous X-linked gene, and one Y-linked gene with no X-linked homologue has lower species-wide diversity than a homologous autosomal copy (SlAp3Y vs. SlAp3A). To test whether this is a general pattern for Y-linked genes, we studied two further recently described X and Y homologous gene pairs in samples from several populations of S. latifolia and S. dioica. Diversity is reduced for both Y-linked genes, compared with their X-linked homologues. Our new data are analysed to show that the low Y effective size cannot be explained by different levels of gene flow for the X vs. the Y chromosomes, either between populations or between these closely related species. Thus, all four Y-linked genes that have now been studied in these plants (the two studied here, and two previously studied genes, have low diversity). This supports other evidence for an ongoing degeneration process in these species.

Non-sex-linked, nuclear cleaved amplified polymorphic sequences in Silene latifolia.

Cleaved amplified polymorphic sequences (CAPS) were identified for five nuclear genes in Silene latifolia. By using published cDNA sequences of S. latifolia, pairs of primers were designed to amplify small regions of six nuclear genes. Targeted regions were successfully amplified, two of which included introns. By using direct sequencing of diploid individuals, suitable polymorphic sites for CAPS markers were rapidly detected in five of six of these gene regions, thus avoiding the tedious screening of a large panel of restriction enzymes. Using controlled progenies, we have also shown that all these CAPS markers segregated independently of the sex phenotype, thus demonstrating that the genes analyzed here are not located in the nonrecombining region of the sex chromosomes.

DNA diversity in sex-linked and autosomal genes of the plant species Silene latifolia and Silene dioica.

The relatively recent origin of sex chromosomes in the plant genus Silene provides an opportunity to study the early stages of sex chromosome evolution and, potentially, to test between the different population genetic processes likely to operate in nonrecombining chromosomes such as Y chromosomes. We previously reported much lower nucleotide polymorphism in a Y-linked gene (SlY1) of the plant Silene latifolia than in the homologous X-linked gene (SlX1). Here, we report a more extensive study of nucleotide diversity in these sex-linked genes, including a larger S. latifolia sample and a sample from the closely related species Silene dioica, and we also study the diversity of an autosomal gene, CCLS37.1. We demonstrate that nucleotide diversity in the Y-linked genes of both S. latifolia and S. dioica is very low compared with that of the X-linked gene. However, the autosomal gene also has low DNA polymorphism, which may be due to a selective sweep. We use a single individual of the related hermaphrodite species Silene conica, as an outgroup to show that the low SlY1 diversity is not due to a lower mutation rate than that for the X-linked gene. We also investigate several other possibilities for the low SlY1 diversity, including differential gene flow between the two species for Y-linked, X-linked, and autosomal genes. The frequency spectrum of nucleotide polymorphism on the Y chromosome deviates significantly from that expected under a selective-sweep model. However, we detect population subdivision in both S. latifolia and S. dioica, so it is not simple to test for selective sweeps. We also discuss the possibility that Y-linked diversity is reduced due to highly variable male reproductive success, and we conclude that this explanation is unlikely.

The spatial structure of sexual and cytonuclear polymorphism in the gynodioecious Beta vulgaris ssp. maritima: I/ at a local scale.

We have analyzed the spatial distribution of the sex phenotypes and of mitochondrial, chloroplast, and nuclear markers within two gynodioecious populations of Beta vulgaris ssp. maritima. Within both populations, sexual phenotype variation is controlled mainly by the cytoplasmic genotype, although in one study population a joint polymorphism of cytonuclear factors is clearly involved. In spite of contrasts in the ecology (mainly due to different habitats), a clear common feature in both populations is the highly patchy distribution of cytoplasmic haplotypes, contrasting with the wide distribution of nuclear diversity. This high contrast between cytoplasmic vs. nuclear spatial structure may have important consequences for the maintenance of gynodioecy. It provides opportunities for differential selection since nuclear restorer alleles are expected to be selected for in the presence of their specific cytoplasmic male sterile (CMS) type, but to be neutral (or selected against if there is a cost of restoration) in the absence of their CMS type. Selective processes in such a cytonuclear landscape may explain the polymorphism we observed at restorer loci for two CMS types.

Effective population sizes for cytoplasmic and nuclear genes in a gynodioecious species. The role of the sex determination system.

Equations are derived for the effective sizes of gynodioecious populations with respect to both nuclear and cytoplasmic genes (N(ec) and N(en), respectively). Compared to hermaphroditism, gynodioecy generally reduces effective population sizes for both kinds of loci to an extent depending on the frequency of females, the sex determination system, and the selfing rate of hermaphrodites. This reduction is due to fitness differences between the sexes and is highly influenced by the mode of inheritance of this fitness. In absence of selfing, nuclear gynodioecy results in a reduction of N(ec) that depends strongly on the dominance of male sterility alleles, while N(en) remains equal to the census number (N). With cytonuclear gynodioecy, both cytoplasmic and nuclear effective sizes are reduced, and at the extreme, dioecy results in the minimum N(ec) values and either minimum or maximum N(en) values (for low or high frequency of females, respectively). When selfing occurs, gynodioecy either increases or decreases N(en) as compared to hermaphroditism with the same selfing rate of hermaphrodites. Unexpectedly, N(ec) also varies with the selfing rate. Thus the genetic sex-determination system appears as a major factor for the nuclear and cytoplasmic genetic diversities of gynodioecious species.

The male-sterility polymorphism of Silene vulgaris: analysis of genetic dat from two populations and comparison with Thymus vulgaris.

Results are given of genetic studies of male sterility using plants from two natural populations from Sussex, England. Both populations have substantial frequencies of females, approximately 0.25 in population 1 and 0.60 in population 3. As in the few other gynodioecious populations studied in detail, many genetic factors are present. In population 1, there are at least two, and more likely three, different cytoplasmic types, one of which appears to produce male sterility in progeny from any hermaphrodite pollen donor; in other words restorer alleles for this cytoplasm are rare or absent from the population. The other two populations can be carried in hermaphrodites that have the dominant restorers. In population 1, there are also probably three restorer loci with complementary recessive male-sterility alleles, as well as a locus with duplicate action, which cannot produce male sterility unless the plant is also homozygous for the recessive allele at another locus. The results from population 3 are quite similar, though there was no evidence in this population for an unrestored sterility cytoplasm. A similar joint nucleocytoplasmic model with multiple restorers fits data from Thymus vulgaris.

Safety, tolerability, and pharmacokinetics of SR 49059, a V1a vasopressin receptor antagonist, after repeated oral administration in healthy volunteers.

UNLABELLED: The conventional evaluation of safety and tolerability during Phase I may not be sufficient for new exploratory non-peptide receptor antagonists as selective vasopressin (AVP) receptor antagonists. Previous research and validation of surrogate markers considerably enhance the understanding of phase I, and may even contribute with high accuracy to an early approach of dose finding. SR 49059 is a new potent and selective non peptide AVP-antagonist, with high affinity, selectivity and efficacy towards both animal and human AVP-V1a receptors. The aim of this study was to assess its tolerability and to determine both its pharmacokinetic and pharmacodynamic profiles. The safety and tolerability of SR 49059 was assessed in an ascending repeated dose tolerability trial, double-blind for each dose. 50 healthy subjects non smoker males, divided into 5 groups (doses) of 10 were included, (8 treated/2 placebo per group) and received oral doses of either 1, 10, 100, 300 or 600 mg of SR 49059 o.d. for 7 days. Clinical tolerability and biological safety was excellent for all subjects up to the highest dose of 600 mg SR 49059 appeared to have no action on AVP plasma level, hemostasis parameters, nor on blood pressure, heart rate, ECG, diuresis or plasma/urine osmolality. Two previously validated surrogate markers using exogenous vasopressin were sufficient to provide evidence of the V1a antagonistic effects of SR 49059 after the first single oral administration, and during the 7 days of treatment: Ex-vivo AVP induced platelet aggregation inhibition: SR 49059 has shown potent antagonistic properties in inhibiting AVP-induced human platelet aggregation in vitro (IC50 = 3.7 nM). Using this ex vivo qualitative test, a dose and time proportional activity was observed at doses as low as 10 mg, and an almost complete inhibition was demonstrated from 100 mg and above, from Day 1 with a steady state level of inhibition from Day 4 up to Day 7. AVP induced blanching skin area inhibition: Intradermic administration of AVP 0.1 ml (25 ng) produced a measurable vasoconstriction (computer analysis of blanching area), which was also dose dependently antagonised by the oral administration of SR 49059 with the same profile as for platelet-aggregation inhibition. Steady state SR 49059 levels were achieved on days 4-5 with moderate (1.8-2.4 fold) accumulation (t1/2: 32 hrs). Cmax values were in the range 0.8-30 ng/ml. The IC50 of AVP (50 nM) -induced platelet aggregation and cutaneous blanching effect were 2.1 +/- 0.7 nM (1.3 ng/mL) and 4.6 +/- 2.5 nM (2.8 ng/mL), respectively. CONCLUSIONS: During early phase I, in addition to the conventional safety profile, validated surrogate markers may provide evidence of activity for selective vasopressin receptor antagonists. The results confirmed that SR 49059 is in human a specific V1a-antagonist without activity at V2 receptors, with a good safety profile.

Pharmacokinetic study of ioxaglate, a low osmolality contrast medium, in patients with renal failure.

Determination of changes in plasma concentration of ioxaglate in patients with renal failure made it possible to demonstrate that distribution and clearance of this contrast medium, as in the case of classic uroangiographic products, conform to an open two-compartment model with clearance from the central compartment. Various pharmacokinetic parameters were calculated. The metabolic clearance of ioxaglate was lower for all 6 patients studied, as compared with results for 3 normal subjects tested, but less marked than with iodamide, a contrast medium involving tubular secretion.

Acute pancreatitis in pregnancy with Ascaris infestation.

Two cases of acute pancreatitis in pregnancy occurred among Vietnamese evacuees in Arkansas. In both cases, Ascaris lumbricoides seemed the likely cause. In endemic areas including the rural southeastern United States, a high index of suspicion for ascariasis is needed because these worms may cause a variety of abdominal disorders including pancreatitis, cholecystitis, and bowel obstruction. In appropriate settings, a therapeutic trial with antihelminthics is indicated.